DNA encoding a novel RG1 polypeptide

ABSTRACT

The present invention relates to novel human extracellular matrix polypeptides, designated RG1, polynucleotides encoding the polypeptides, methods for producing the polypeptides, expression vectors and genetically engineered host cells for expression of the polypeptides. The invention further relates to methods for utilizing the polynucleotides and polypeptides in research, diagnosis, and therapeutic applications.

This application claims the benefit of U.S. Provisional Application No.60/172,370, filed Dec. 16, 1999, which is incorporated herein in full byreference.

FIELD OF THE INVENTION

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; methods of making the polynucleotides and polypeptides,and their variants and derivatives; antibodies directed toward thepolypeptides, their variants and derivatives; and uses of thepolynucleotides, polypeptides, variants, derivatives and antibodies. Inparticular, in these and in other regards, the invention relates tonovel human extracellular matrix polypeptides (designated RG1),polynucleotides which encode these polypeptides, antibodies directedtoward these polypeptides, and antisense polynucleotides that block RG1expression.

BACKGROUND OF THE INVENTION

Prostate cancer is a frequently occurring disease in man, in that it isfound in about one third of men over the age of 45. There is evidencefor both genetic and environmental causes, with the majority of casesprobably being the result of a combination of both factors. Studies offamilial cancer have suggested that genetic predisposition plays a rolein about 5-10% of all prostate cancers, and in about 45% of cases in menyounger than 55.

There is evidence that prostate cancer develops as a multi-step disease,with one of the precursor lesions being prostatic intraepithelialneoplasia (PIN). Early stages of the disease are androgen dependent,while later stages are hormone independent. A proliferative disorder ofthe prostate known as benign prostatic hyperplasia is often detectedclinically but is probably not a stage in the development of cancer. Itis, however, frequently associated with prostate cancer. Cancers in theprostate are often multifocal, generally slow growing, andheterogeneous. Late stage cancers frequently metastasize to the lymphnodes and to the bone.

Prostate cancer is usually diagnosed by physical examination and byserum levels of prostate specific antigen (PSA). Radical prostatectomyis the treatment of choice for localized disease. Advanced metastaticdisease is treated currently by androgen ablation induced by orchiectomyor treatment with GnRH (gonadotrophin releasing hormone), and byanti-androgen therapy. However, advanced disease almost invariablybecomes hormone resistant and there is no cure for progressive disease.Moreover, there are serious side effects associated with both radicalprostatectomy and androgen ablation therapy. These include a high riskof incontinence and impotence associated with radical prostatectomy andbone fractures and osteoporosis associated with androgen ablationtherapy.

There is, therefore, a considerable need for new therapeutic approachesfor both early and late stage prostate cancer. There is also asignificant need for new diagnostic agents, in particular agents thatcan discriminate stages of the disease, as this significantly influencesthe treatment options. For example, if disease has progressed beyond theprostate and has metastasized to the lymph nodes, radical prostatectomyis not undertaken as it has no effect on progression, but may havesignificant unwanted side effects. An agent that could detectmetastasis, in vivo, would have considerable value.

Changes in the expression of specific proteins have been demonstrated inprostate cancer including abnormal p53 expression in late stage prostatecancer, reduced levels of TGF-β receptors, reduced levels of E-cadherin,C-Cam (a cell adhesion molecule), and several integrins. The expressionof the oncogene bcl-2 is strikingly elevated in late stage androgenindependent tumors, and prognosis for patients expression bcl-2 atelevated levels is relatively poor. While the previously mentionedchanges in gene expression are well documented, no changes in expressionhave been identified that have been demonstrated to be causative for thedisease. It would, therefore, be useful to identify new proteins whoseexpression is linked to the presence or development of prostate tumorswhich could serve as molecular targets for prostate cancer diagnosis andtherapy.

This invention discloses a new homologue to a superfamily ofextracellular matrix proteins. This homologue, named RG1 is expressed inprostate tissue and may be over-expressed in prostate tumors.

The extracellular matrix is a complex meshwork of collagen and elastin,embedded in a viscoelastic ground substance composed of proteoglycansand glycoproteins. The matrix exists as a three dimensional supportingscaffold that isolates tissue compartments, mediates cell attachment anddetermines tissue architecture (Bissel et al., J. Theor. Biol. 99:31-68,1982; Carlson et al., Proc. Natl. Acad. Sci. USA 78:2403-2406, 1981).The matrix acts as a macromolecular filter (Hay, E. D., Cell Biology ofExtracellular Matrix, New York, Plenum Press, 1982) and also influencescytodifferentiation, mitogenesis, and morphogenesis (Gospodarowiczs, D.,Cancer Res. 38:4155-171, 1978). The biochemical interactions betweennormal cells and the matrix may be altered in neoplasia, and this mayinfluence tumor proliferation. Tumor cells can interact with the matrixin different ways. First, tumor cells can attach to the matrix viaspecific plasma membrane receptors (Terranova et al., Cancer Res.42:2265-2269, 1982). Second, degradation of the matrix is mediated by acascade of enzymes that are contributed by the tumor cell and the host(Eisen et al., Bioch. Biophys. Acta 151:637-645, 1968). Third, indifferentiated areas of the tumor, tumor cells may synthesize andaccumulate matrix or induce the host cell to accumulate excessive matrix(Brownstein et al., Cancer 40: 2979-2986, 1977).

RG1 shows homology to a superfamily of extracellular matrix proteins,encoded by the Mindin/F-spondin genes. The gene family is united by twoconserved spondin domains, FS1 and FS2, near the amino terminus and atleast one thrombospondin type 1 repeat (TSR1) at the carboxy terminus(Shimeld, S. M., Mol. Biol. Evol. 15(9): 1218-1223, 1998). The TSR motifwas originally found in the vertebrate extracellular matrix proteins(Bornstein, P., J. Cell Biol. 130:503-506, 1995) and has subsequentlybeen found in several other extracellular matrix proteins. There areseveral lines of evidence that TSR's mediate cell adhesion and play akey role in tumorigenesis. For example, it has been demonstrated thatproteolytic fragments of thrombospondin that contain the TSR's, andsynthetic peptides having sequences corresponding to the TSR region ofthrombospondin, promote tumor cell adhesion and metastasis (Prater etal., J. Cell Biol. 112:1031-1040, 1991; Tuszynski and Nicosia, BioEssays18:71-76, 1996), have anti-angiogenic activity (Tolsma et al., J. CellBiol. 122:497-511, 1993) and inhibit platelet aggregation and melanomametastasis (Tuszynski et al., J. Cell Biol. 116:209-217, 1992).

Currently, the members of this superfamily include a gene inCaenorhabditis elegans, a single gene in Drosophila and multiple genesin vertebrates. In C. elegans, the gene F10E7.4 encodes for five TSR'sin addition to the FS1 and FS2 domains (Higashijima et.al., Dev. Biol.192:211-227, 1997). In Drosophila, the family member termed M-spondin(mspo) contains the FS1 and FS2 domains and a single TSR (Umemiya etal., Dev. Biol. 186:165-176, 1997). The M-spondin gene encodes asecreted protein that is localized at the muscle attachment sites andseems to function as an extracellular matrix protein that supportsmuscle—apodeme attachment. The family members in vertebrates includegenes isolated from zebrafish (Mindin1 and Mindin2, F-spondin1, andF-spondin2), rat F-spondin, Xenopus F-spondin and rat Mindin. Mindin1and Mindin2 are closely related to each other and have a gene structuresimilar to that of Drosophila M-spondin. Both Mindin1 and Mindin2 genesencode for a single TSR in addition to the FS1 and FS2 domains(Higashijima et.al., Dev. Biol. 192:211-227, 1997). Zebrafish F-spondinland F-spondin2, rat F-spondin (Klar et al., Cell 69:95-110, 1992) andXenopus F-spondin (Altaba et al., Proc. Natl. Acad. Sci. USA90:8268-8272) genes all have similar structures, encoding six copies ofthe TSR's in addition to the FS1 and FS2 domains. In vertebrates, theMindin/F-spondin superfamily can be classified into two groups: thosegenes closely related to the original rat F-spondin and Mindin genes andthose genes closely related to the Drosophila M-spondin gene. Bothvertebrate Mindin and F-spondin genes code for proteins that areprimarily expressed by the floor plate of the neural tube duringembryonic development.

Recently, a single F-spondin related gene, AmphiF-spondin, has beenisolated from amphioxus (Shimeld, S. M., Mol. Biol. Evol. 15(9):1218-1223,1998). Based on molecular phylogenetics, AmphiF-spondin isclosely related to a particular subgroup of vertebrate F-spondin genesthat encode six TSR's. AmphiF-spondin encodes three TSR's and twofibronectin type III repeats, one of which has strong identity to afibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Theexpression of the protein is found through most of the central nervoussystem and is not confined to the midline as described for thevertebrate Mindin and F-spondin proteins.

These data suggest that extracellular matrix proteins, such as the novelRG1 protein, which is a homologue of the Mindin/F-spondin superfamily,would be good candidates for use in diagnosis of cancer and therapeuticintervention.

SUMMARY OF THE INVENTION

The present invention provides a polynucleotide sequence which uniquelyencodes a novel protein designated herein as RG1. The RG1 polypeptideshows homology to the rat Mindin extracellular matrix protein. Itcontains a hydrophobic signal sequence at the N-terminus, two spondindomains (FS1 and FS2), and a thrombospondin type 1 repeat at itsC-terminus. RG1 shows 89.7% similarity to rat Mindin. The polynucleotidesequence, designated herein as rg1, and described herein in FIG. 1 (SEQID NO. 1), encodes the amino acid sequence for RG1, which is shown inFIG. 2 (SEQ ID NO. 2).

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelproteins with homology to the Mindin family of extracellular matrixproteins, as shown by comparison of the amino acid sequence set out inFIG. 2 (SEQ ID NO: 2) and known amino acid sequences of otherextracellular matrix proteins.

It is a further object of the invention, moreover, to providepolynucleotides that encode such polypeptides, particularlypolynucleotides that encode the polypeptide designated herein as RG1.

In accordance with this aspect of the invention there are providedisolated polynucleotides encoding RG1, including mRNAs, cDNAs, and, infurther embodiments of this aspect of the invention, biologically,diagnostically, clinically or therapeutically useful variants, analogsor derivatives thereof, or fragments thereof, including fragments of thevariants, analogs and derivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of polynucleotidesthat encode variants of the polypeptide designated herein as RG1.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as RG1 as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of RG1 encoded by naturally occurring allelicvariants of the rg1 polynucleotide.

It is another object of the invention to provide a method of producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods of producing the aforementioned RG1polypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived RG1-encoding polynucleotideunder conditions for expression of human RG1 in the host and thenrecovering the expressed polypeptide.

In accordance with another object of the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for inter alia research,biological, clinical and therapeutic purposes.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for assessing RG1 expression in cells by determining RG1polypeptides or RG1-encoding mRNA; and assaying genetic variation andaberrations, such as defects, in rg1 genes.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize to rg1sequences.

It is a further object of the invention to provide antibodies which arehighly selective for RG1 polypeptides, or fragments thereof, and whichmay be employed in a method for diagnosis and/or detection of RG1expression, which may be associated with prostate cancer. In accordancewith certain preferred embodiments of this aspect of the invention,antibodies are labeled in such a way as to produce a detectable signal.Particularly preferred would be an antibody labeled with a radiolabel,an enzyme, a chromophore or a fluorescer.

In a further aspect of the invention there are provided antibodies whichare conjugated to a therapeutic agent for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. Particularly preferred in this regard are therapeutic agentswhich are cytotoxic. In certain preferred embodiments in this regard isadministration of such conjugated antibodies to a human patient fortreatment of a disease state characterized by RG1 activity or expressionsuch as prostate cancer.

In a further aspect of the invention, peptides and anti-idiotypicantibodies are provided which can be used to stimulate an immuneresponse.

In a further aspect of the invention there are provided ribozymes andpolynucleotides complementary to rg1 polynucleotides (i.e. antisensepolynucleotides) for administration to cells in vitro, to cells ex vivoand to cells in vivo, or to a multicellular organism. Particularlypreferred in this regard is administration of antisense molecules to ahuman patient for treatment of a disease state, such as prostate canceror benign prostatic hyperplasia, which is alleviated by decreasing thelevel of RG1 activity.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Polynucleotide sequence of rg1 (SEQ ID NO: 1), which encodes thebiologically or immunologically active form of RG1.

FIG. 2: Deduced amino acid sequence of RG1 (SEQ ID NO: 2), with theF-spondin domains single underlined, and the thrombospondin domaindouble underlined.

FIG. 3: Amino acid alignment of RG1 (SEQ ID NO: 2) with the sequence ofrat Mindin (SEQ ID NO: 13). The sequence of RG1 (SEQ ID NO: 2) is on thetop.

FIG. 4: Polynucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO:2) sequences of RG1.

FIG. 5: Expression of rg1 mRNA in human tissues by Taqman based PCRanalysis. RNA from human tissues, both tumor and normal, was isolated bystandard techniques. Primers and probe to detect rg1 mRNA expressionwere designed using Perkin Elmer's Primer Express software andsynthesized by Synthetic Genetics. Rg1 mRNA was detected in humanprostate tissues. A much lower expression of rg1 mRNA could be detectedin other tissues, e.g. liver.

FIG. 6: Purification of native RG1 protein secreted by LNCaP cells.Western blot analysis, using antisera generated against a synthetic RG1peptide sequence (3C, SEQ ID NO: 10; see Example 4), to detect nativeRG1 protein secreted from LNCaP cells. Elution fractions fromQ-Sepharose chromatography of concentrated LNCaP cell conditioned media:(L) column load, (F) column flow-thru, (1-12) elution fractions acrosssalt gradient. The predicted molecular weight of RG1 is ˜36 kD, howeverthe bacterially expressed RG1, BHK-expressed RG1 and the LNCaP-expressedRG1 protein all have been observed to migrate at ˜45 kD on PAGE (L,fractions 6-9).

FIG. 7: Immunohistochemical staining of RG1 expression in human prostatetissues. Prostate tissues were obtained from the Urology Department atStanford University School of Medicine. The staining was carried outusing the Vector ABC-AP kit (AK5002). Staining was visualized with aVector Red substrate kit (SK-5100) and counterstained with Hematoxylin.The results show strong peri-luminal membrane staining in glandformations.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification, examples and appended claims, unlessspecified to the contrary, the following terms have the meaningindicated.

“RG1” refers to the polypeptide having the amino acid sequence set outin FIG. 2 (SEQ ID NO: 2); variants, analogs, derivatives and fragmentsthereof, and fragments of the variants, analogs and derivatives. Theterms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 2 (SEQ ID NO: 2) mean a polypeptide which retainsessentially the same biological and/or immunological activity as thepolypeptide of FIG. 2 (SEQ ID NO: 2).

“rg1” refers to the polynucleotide having the sequence set out in FIG. 1(SEQ ID NO: 1) and polynucleotides encoding polypeptides having theamino acid sequence of RG1 set out in FIG. 2 (SEQ ID NO: 2); and topolynucleotides encoding RG1 variants, analogs, derivatives andfragments, and fragments of the variants, analogs and derivatives. Rg1also refers to such polynucleotides composed of RNA as well as topolynucleotides which are the complement of polynucleotides which encodethe polypeptide sequence set out in FIG. 2 (SEQ ID NO: 2).

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term “polynucleotide” includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritium-labelled bases, to name just two examples, arepolynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term “polynucleotide” as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

“Polypeptides”, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins of which there are manytypes.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. Among the known modifications which may be presentin polypeptides of the present invention are, to name an illustrativefew, acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a polynucleotide or polynucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, I. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W. H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Seifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationalevents, including natural processing events and events brought about byhuman manipulation which do not occur naturally Circular, branched andbranched circular polypeptides may be synthesized by non-translationalnatural processes and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentto the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

“Polynucleotide encoding a polypeptide” as used herein encompassespolynucleotides which include a sequence encoding a polypeptide of thepresent invention, particularly the RG1 polypeptide having the aminoacid sequence set out in FIG. 2 (SEQ ID NO: 2). The term encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by introns)together with additional regions.

“Biological activity” refers to the structural, regulatory orbiochemical functions of naturally occurring RG1 polypeptide.

“Immunologic activity” refers to the capability of the natural,recombinant or synthetic RG1, or any fragment thereof, to induce aspecific immune response in appropriate animals or cells and to bindwith specific antibodies.

“Oligonucleotide(s)” refers to relatively short polynucleotides. Oftenthe term refers to single-stranded deoxyribonucleotides but it can referas well to single- or double-stranded ribonucleotides, RNA DNA hybridsand double-stranded DNAs, among others. Oligonucleotides, such assingle-stranded DNA probe oligonucleotides, often are synthesized bychemical methods, such as those implemented on automated oligonucleotidesynthesizers. However, oligonucleotides can be made by a variety ofother methods, including in vitro recombinant DNA-mediated techniquesand by expression of DNAs in cells and organisms. “Oligonucleotides” or“oligomers” or polynucleotide “fragment”, “portion”, or “segment” refersto a polynucleotide sequence of at least about 10 nucleotides and asmany as about 60 nucleotides, preferably about 15 to 30 nucleotides, andmore preferably about 20-25 nucleotides.

“Naturally occurring RG1” refers to RG1 produced by human cells thathave not been genetically engineered and specifically contemplatesvarious RG1 forms arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation, acylation, and cleavage.

“Variant(s)” of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

(1) A polynucleotide that differs in polynucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the polynucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical.

As noted below, changes in the polynucleotide sequence of the variantmay be silent. That is, they may not alter the amino acids encoded bythe polynucleotide. Where alterations are limited to silent changes ofthis type a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thepolynucleotide sequence of the variant may alter the amino acid sequenceof a polypeptide encoded by the reference polynucleotide. Suchpolynucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions, fusions and truncations, which may be present in anycombination. Recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide, tochange ligand-binding affinities, interchain affinities, or polypeptidedegradation or turnover rate.

“Allelic variant” refers to an alternative form of the rg1polynucleotide. Alleles result from a mutation, i.e., a change in thepolynucleotide sequence, and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one or many allelic forms. Common mutationalchanges which give rise to alleles are generally ascribed to naturaldeletions, additions or substitutions of nucleotides. Each of thesetypes of changes may occur alone, or in combination with the others, orone or more times in a given sequence.

“Derivative” refers to polynucleotides or polypeptides derived fromnaturally occurring rg1 or RG1, respectively, by chemical modificationssuch as ubiquitination, labeling (e.g., with radionuclides, variousenzymatic modifications), pegylation (derivatization with polyethyleneglycol) or by insertion or substitution of amino acids such as ornithine(or substitution of the nucleotides which code for such as an aminoacid), which do not normally occur in human proteins.

“Deletion” is defined as a change in either polynucleotide or amino acidsequences in which one or more polynucleotides or amino acid residues,respectively, are absent.

“Insertion” or “addition” is that change in a polynucleotide or aminoacid sequence which has resulted in the addition of one or morepolynucleotides or amino acid residues, respectively, as compared to thenaturally occurring polynucleotide or amino acid sequence.

“Substitution” results from the replacement of one or morepolynucleotides or amino acids by different polynucleotides or aminoacids, respectively.

Preferably, amino acid substitutions are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine, i.e. conservative amino acid replacement. Insertions ordeletions are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in thepolypeptide using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

“Fragment” is a polypeptide having an amino acid sequence that entirelyis the same as part but not all of the amino acid sequence of theaforementioned RG1 polypeptides and variants or derivatives thereof.

A polypeptide “fragment”, “portion”, or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and invarious embodiments, at least about 17 or more amino acids.

“Recombinant” or “recombinant DNA molecule” refers to a polynucleotidesequence which is not naturally occurring, or is made by the artificialcombination of two otherwise separated segments of sequence. By“recombinantly produced” is meant artificial combination oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of polynucleotides, e.g., by geneticengineering techniques. Such manipulation is usually done to replace acodon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a sequence recognitionsite. Alternatively, it is performed to join together polynucleotidesegments with desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonnatural forms. Restriction enzyme recognition sites, regulationsequences, control sequences, or other useful features may beincorporated by design. “Recombinant DNA molecules” include cloning andexpression vectors. “Recombinant” may also refer to a polynucleotidewhich encodes a polypeptide and is prepared using recombinant DNAtechniques.

“Isolated” means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living animal inits natural state is not “isolated”, but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs. Polynucleotidesand polypeptides may occur in a composition, such as media formulations,solutions for introduction of polynucleotides or polypeptides, forexample, into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

“Substantially pure” and “substantially homogenous” are usedinterchangeably and describe RG1 polypeptide, or fragments thereof, or apolynucleotide segment encoding same, where such polypeptide orpolynucleotide is separated from components that naturally accompany it.An RG1 polypeptide or fragment thereof, or DNA segment encoding same issubstantially free of naturally-associated components when it isseparated from the native contaminants which accompany it in its naturalstate. Thus, a polypeptide that is chemically synthesized or synthesizedin a cellular system different from the cell in which it naturallyoriginates will be substantially free from its naturally-associatedcomponents. Similarly, a polynucleotide that is chemically synthesizedor synthesized in a cellular system different from the cell in which itnaturally originated will be substantially free from itsnaturally-associated components.

“Homologous”, when used to describe a polynucleotide, indicates that twopolynucleotides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least 70% of the nucleotides, usually from about 75% to99%, and more preferably at least about 98 to 99% of the nucleotides.

“Similarity”, when used to describe a polypeptide, is determined bycomparing the amino acid sequence and the conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide

“Polymerase chain reaction” or “PCR” refers to a procedure whereinspecific pieces of DNA are amplified as described in U.S. Pat. No.4,683,195, issued Jul. 28, 1987. Generally, sequence information fromthe ends of the polypeptide fragment of interest or beyond needs to beavailable, such that oligonucleotide primers can be designed; theseprimers will point towards one another, and will be identical or similarin sequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers will coincide with the ends ofthe amplified material. PCR can be used to amplify specific DNAsequences from total genomic DNA, cDNA transcribed from total cellularRNA, plasmid sequences, etc. (See generally Mullis et al., Cold SpringHarbor Symp. Quant Biol., 51: 263, 1987; Erlich, ed., PCR Technology,Stockton Press, NY, 1989).

“Stringency” typically occurs in a range from about T_(m) (meltingtemperature)-5° C. (5° below the T_(m) of the probe) to about 20° C. to25° C. below T_(m). As will be understood by those of skill in the art,a stringent hybridization can be used to identify or detect identicalpolynucleotide sequences or to identify or detect similar or relatedpolynucleotide sequences. As herein used, the term “stringentconditions” means hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences.

“Hybridization” as used herein, shall include “any process by which apolynucleotide strand joins with a complementary strand through basepairing” (Coombs, J., Dictionary of Biotechnology, Stockton Press, NewYork, N.Y., 1994).

“Therapeutically effective dose” refers to that amount of polypeptide orits antibodies, antagonists, or inhibitors, including antisensemolecules and ribozymes, which ameliorate the symptoms or conditions ofa disease state. Therapeutic efficacy and toxicity of such compounds canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.

“Treating” or “treatment” as used herein covers the treatment of adisease-state in a human patient, which disease-state is associated withprostate tumor growth and includes disease states in which the patientis in need of decreased levels of RG1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel RG1 polypeptides, rg1polynucleotides, and antibodies directed toward RG1 polypeptides, amongother things, as described in greater detail below. In particular, theinvention relates to novel RG1 polypeptides and the polynucleotidesencoding these RG1 polypeptides, and relates especially to RG1 havingthe amino acid sequence set out in FIG. 2 (SEQ ID NO: 2) and rg1 havingthe polynucleotide sequence set out in FIG. 1 (SEQ ID NO: 1). Thepresent invention also encompasses RG1 variants. A preferred RG1 variantis one having at least 70% similarity (preferably at least 70% identity)to the polypeptide sequence shown in FIG. 2 (SEQ ID NO: 2) and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide shown in FIG. 2 (SEQ ID NO: 2) and stillmore preferably at least 95% similarity (still more preferably at least95% identity) to the polypeptide sequence shown in FIG. 2 (SEQ ID NO: 2)and also includes portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The coding sequence for the predicted RG1 polypeptide begins 296 basepairs from the 5′ end of the nucleotide sequence shown in FIG. 1 (SEQ IDNO: 1). RG1 contains three structural domains characteristic ofMindin/F-spondin superfamily of extracellular matrix proteins: twospondin domains (FS1 and FS2), comprising amino acids 31 to 103 and 138to 221, respectively, and a thrombospondin domain, comprising aminoacids 278 to 330.

The present invention is based in part on the structural homology shownin FIG. 3 between RG1 (SEQ ID NO: 2) and rat Mindin (SEQ ID NO: 13),another member of the extracellular matrix protein family. The aminoacid sequence of RG1 (SEQ ID NO: 2) is approximately 89.7% similar torat Mindin (SEQ ID NO: 13).

The present invention is also based in part on the expression profile ofRG1, as demonstrated by its expression in prostate tissue libraries andover-expression in prostate tumor libraries. This tissue profile is seenin analysis of mRNA expression in tissue samples from normal and tumortissues by PCR-based Taqman analysis. This method of analysisdemonstrated that mRNA encoding RG1 is over-expressed in prostatetissues as compared with other tissues.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides that encode the RG1 polypeptide havingthe deduced amino acid sequence of FIG. 2 (SEQ ID NO: 2).

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 (SEQ ID NO: 1), a polynucleotide of thepresent invention encoding a RG1 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA from cells of human tissue as starting material.Illustrative of the invention, the polynucleotide sequence in FIG. 1(SEQ ID NO: 1) was found in cDNA clones obtained from human prostatetissues. Rg1 was identified as a gene expressed in the prostate bymining Incyte's LifeSeq database. The nucleotide sequence was identifiedby an annotation search of the database, using the “Protein Function”tool provided by Incyte for the purpose of searching the database. Thenucleotide sequence was found in the category of cell adhesion moleculesin the annotated database and was described as a homologue of f-spondinElectronic Northern analysis of the distribution of rg1 polynucleotidesequences in the set of libraries in the database revealed that rg1 wasexpressed at high levels in the prostate libraries and at lower levelsin a number of other tissue libraries, including those from normal andtumor tissues.

Following assembly of the set of rg1 clones in the database into acontiguous polynucleotide sequence, and editing of the contiguoussequence, a full-length coding sequence was identified in the predictedassembled polynucleotide. This sequence coded for a protein withhomology to rat mindin.

Incyte clones 1640796, 1712252, and 1880265 were obtained from Incytefor experimental work and clone 3360733 was identified as containing themost 5′ nucleotide sequence. This clone was fully sequenced andcontained the full coding sequence for the predicted RG1 protein. Thissequence is shown in FIG. 1 (SEQ D NO: 1).

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof, or by methods described herein.The DNA may be double-stranded or single-stranded. Single-stranded DNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also referred to as the anti-sense strand.

The sequence which encodes the polypeptide may be identical to thecoding sequence of the polynucleotide shown in FIG. 1 (SEQ ID NO: 1). Italso may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2 (SEQ ID NO: 2).

Polynucleotides of the present invention which encode the polypeptide ofFIG. 2 (SEQ ID NO: 2) may include, but are not limited to, the codingsequence for the polypeptide itself; the coding sequence of thepolypeptide, together with additional, non-coding sequences, includingfor example, but not limited to, introns and non-coding 5′ and 3′sequences, such as the transcribed, non-translated sequences that play arole in transcription, mRNA processing (for example, splicing andpolyadenylation signals) or additional coding sequences which code foradditional amino acids, such as those which provide additionalfunctionalities. Thus, for instance, the polypeptide may be fused to amarker sequence, such as a peptide, which facilitates purification ofthe fused polypeptide. In certain preferred embodiments of this aspectof the invention, the marker sequence is a hexa-histidine peptide, suchas the tag provided in a pTrcHisB vector (Invitrogen, Carlsbad, Calif.)among others, many of which are commercially available. As described inGentz et al. (Proc. Natl. Acad. Sci., USA 86: 821-824, 1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein.

The polynucleotides may encode a polypeptide which is the polypeptideplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the polypeptide (when the active form has more than onepolypeptide chain, for instance). Such sequences may play a role inprocessing of a polypeptide from precursor to final form, may facilitatepolypeptide trafficking, may prolong or shorten polypeptide half-life ormay facilitate manipulation of a polypeptide for assay or production,among other things. As generally is the case in situ, the additionalamino acids may be processed away from the polypeptide by proteolyticenzymes.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2 (SEQ ID NO: 2). A variant of the polynucleotide may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by polynucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more polynucleotides. The variants may be altered incoding or non-coding regions or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of RG1 set out in FIG. 2 (SEQ ID NO: 2); variants, analogs,derivatives and fragments thereof, and fragments of the variants,analogs and derivatives

Further particularly preferred in this regard are polynucleotidesencoding RG1 variants, analogs, derivatives and fragments, and variants,analogs and derivatives of the fragments, which have the amino acidsequence of the RG1 polypeptide of FIG. 2 (SEQ ID NO: 2) in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions and deletions,which do not alter the properties and activities of the RG1 polypeptide.Also especially preferred in this regard are conservative substitutions.Most highly preferred are polynucleotides encoding polypeptides havingthe amino acid sequence of FIG. 2 (SEQ ID NO. 2) without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding the RG1polypeptide having the amino acid sequence set out in FIG. 2 (SEQ ID NO:2), and polynucleotides which are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical to a polynucleotide encoding theRG1 polypeptide and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these, those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological activity as the polypeptide encoded by thepolynucleotide sequence of FIG. 1 (SEQ ID NO: 1).

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probes for cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding RG1 and to isolatecDNA and genomic clones of other genes that have a high sequencesimilarity to the rg1 gene Such probes generally will comprise at least15 bases. Preferably, such probes will have at least 30 bases and mayhave at least 50 bases.

For example, the coding region of the rg1 gene may be isolated byscreening libraries using synthetic oligonucleotide probes that havebeen designed using the known DNA sequence. For example, a labeledoligonucleotide having a sequence complementary to that of apolynucleotide of the present invention can be used to screen a libraryof cDNA or genomic DNA to identify clones that hybridize to the probe.

In sum, a polynucleotide of the present invention may encode apolypeptide, a polypeptide plus a leader sequence (which may be referredto as a prepolypeptide).

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the polypeptide fragments, polynucleotides thathybridize to polynucleotides encoding polypeptide fragments,particularly those that hybridize under stringent conditions, andpolynucleotides, such as PCR primers, for amplifying polynucleotidesthat encode polypeptide fragments. In these regards, preferredpolynucleotides are those that correspond to preferred polypeptidefragments, as discussed below.

Polypeptides

The present invention further relates to a RG1 polypeptide which has thededuced amino acid sequence of FIG. 2 (SEQ ID NO: 2).

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms fragment, derivative and analog whenreferring to the polypeptide of FIG. 2 (SEQ ID NO: 2) means apolypeptide which retains essentially the same biological activity assuch a polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments, it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 2 (SEQ IDNO: 2) may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol) or (iv) one in which theadditional amino acids are fused to the polypeptide, such as a leader orsecretory sequence or a sequence which is employed for purification ofthe polypeptide. Such fragments, derivatives and analogs are deemed tobe within the scope of those skilled in the art from the teachingsherein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of RG1 set out inFIG. 2 (SEQ ID NO: 2), variants, analogs, derivatives and fragmentsthereof, and variants, analogs and derivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile, interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the RG1 polypeptide of FIG.2 (SEQ ID NO: 2) in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the RG1 polypeptide. Also especially preferred in thisregard are conservative substitutions. Most highly preferred arepolypeptides having the amino acid sequence of FIG. 2 (SEQ ID NO: 2)without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention also include the polypeptideof FIG. 2 (SEQ ID NO: 2) as well as polypeptides which have at least 70%similarity (preferably at least 70% identity) to the polypeptide of FIG.2 (SEQ ID NO: 2) and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 2 (SEQ IDNO: 2) and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of FIG. 2 (SEQ IDNO: 2) and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptides bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of RG1, most particularlyfragments of the RG1 of FIG. 2 (SEQ ID NO: 2), and fragments of variantsand derivatives of the RG1 of FIG. 2 (SEQ ID NO: 2).

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned RG1 polypeptides and variants or derivativesthereof.

Such fragments may be “free-standing,” i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of an RG1 polypeptideof the present invention comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre- andpropolypeptide regions fused to the amino terminus of the RG1 fragmentand an additional region fused to the carboxyl terminus of the fragment.Therefore, fragments in one aspect of the meaning intended herein,refers to the portion or portions of a fusion polypeptide or fusionprotein derived from RG1.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 25 to about 331 aminoacids.

In this context “about” includes the particularly recited range andranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes. For instance, about 331 aminoacids in this context means a polypeptide fragment of 25 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 331 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 25minus several amino acids to 331 plus several amino acids to as narrowas 25 plus several amino acids to 331 minus several amino acids.

Highly preferred in this regard are the recited ranges plus or minus asmany as 5 amino acids at either or at both extremes. Particularly highlypreferred are the recited ranges plus or minus as many as 3 amino acidsat either or at both the recited extremes. Especially particularlyhighly preferred are ranges plus or minus 1 amino acid at either or atboth extremes or the recited ranges with no additions or deletions Mosthighly preferred of all in this regard are fragments from about 25 toabout 331 amino acids.

Among especially preferred fragments of the invention are truncationmutants of RG1. Truncation mutants of RG1 include variants orderivatives of the sequence of FIG. 2 (SEQ ID NO: 2), except fordeletion of a continuous series of residues (that is, a continuousregion, part or portion) that includes the amino terminus of thesequence shown in FIG. 2 (SEQ ID NO: 2), or a continuous series ofresidues that includes the carboxyl terminus or, as in double truncationmutants, deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Fragmentshaving the size ranges set out above also are preferred embodiments oftruncation fragments, which are especially preferred among fragmentsgenerally.

Especially preferred in this aspect of the invention are fragmentscharacterized by biological and/or immunological attributes of RG1. Suchfragments include those containing the predicted structural domains ofRG1, which encompass at least amino acid 31 to 103, 138 to 221 and 278to 330 or those fragments used to generate antibodies, such as thosedescribed in Example 4.

Certain preferred regions in these regards are set out in FIG. 2 (SEQ IDNO: 2), and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequenceset out in FIG. 2 (SEQ ID NO: 2).

Among highly preferred fragments in this regard are those that compriseregions of RG1 that combine several structural features, such as thefeatures set out above. In this regard, the two spondin and onethrombospondin domains, encompassing about amino acids 31 to 103, 138 to221, and 278 to 330, respectively, which are characteristic of theMindin/spondin superfamily of extracellular matrix proteins, areespecially preferred regions. Such regions may be comprised within alarger polypeptide or may be by themselves a preferred fragment of thepresent invention, as discussed above. It will be appreciated that theterm “about” as used in this paragraph has the meaning set out aboveregarding fragments in general.

Further preferred regions are those that mediate activities of RG1. Mosthighly preferred in this regard are fragments that have a chemical,biological or other activity of RG1, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Highly preferred in this regard are fragments that containregions that are homologs in sequence, or in position, or in bothsequence and position to active regions of related polypeptides, such asthe other proteins of the Mindin family, which includes RG1.

Vectors, Host Cells, and Expression Systems

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Suchtechniques are described in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 andAusubel, F. M. et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., 1989.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case, thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques. In accordance with thisaspect of the invention, the vector may be, for example, a plasmidvector, a single or double-stranded phage vector, a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors, also may be and preferably are introduced intocells as packaged or encapsidated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives A variety of vectors suitable to this aspect of the invention,including constitutive and inducible expression vectors for use inprokaryotic and eukaryotic hosts, are well known and employed routinelyby those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter al/ia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, retroviruses, and alphavirues such as Sindbis virus, andvectors derived from combinations thereof, such as those derived fromplasmid and bacteriophage genetic elements such as cosmids andphagemids, all may be used for expression in accordance with this aspectof the present invention. Generally, any vector suitable to maintain,propagate or express polynucleotides to express a polypeptide in a hostmay be used for expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseof skill, are set forth in great detail in Sambrook et al. citedelsewhere herein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp, tac, and trcpromoters, the SV40 early and late promoters and promoters of retroviralLTRs to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention, are well known and may readily be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the transcriptsexpressed by the constructs will include a translation initiating AUG atthe beginning and a termination codon appropriately positioned at theend of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase, neomycin,puromycin, or hygromycin resistance for eukaryotic cell culture, andtetracycline, theomycin, kanamycin or ampicillin resistance genes forculturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli. Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells, and plant cells, preferably insectcells BTI-TN-5B1-4. Hosts for a great variety of expression constructsare well known, and those of skill will be enabled by the presentdisclosure readily to select a host for expressing a polypeptides inaccordance with this aspect of the present invention.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-7lines of monkey kidney fibroblast (Gluzman et al., Cell 23: 175, 1991).Other cell lines capable of expressing a compatible vector include forexample, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the polynucleotide sequence coding for RG1 may be ligated intoan adenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome will result in a viable virus capableof expressing RG1 in infected host cells (Logan and Shenk, Proc. Natl.Acad. Sci. USA 81:3655-59, 1984). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE7O,pQE6O and pQE-9, available from Qiagen USA (Valencia, Calif.), pBSvectors, Phagescript® vectors, Bluescript® vectors, pNH8A, pNHI6a,pNHI8A, pNH46A, available from Stratagene (LaJolla, Calif.); andptrc99a, pK223-3, pKK233-3, pDR540, pRIT5 available from PharmaciaBiotech (Piscataway, N.J.). Most preferred is the pTrcHisB vector,available from Invitrogen. Among preferred eukaryotic vectors arepWLNEO, pSV2CAT, pOG44, PXTI and pSG available from Stratagene; andPSVK3, pBPV, pMSG and pSVL available from Pharmacia Biotech. Mostpreferred is the pClneo vector available from Promega. These vectors arelisted solely by way of illustration of the many commercially availableand well known vectors that are available to those of skill in the artfor use in accordance with this aspect of the present invention. It willbe appreciated that any other plasmid or vector suitable for, forexample, introduction, maintenance, propagation or expression of apolynucleotide or polypeptide of the invention in a host may be used inthis aspect of the invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“cat”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-B and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lad and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters, the trppromoter, and the trc hybrid promoter, which is derived from the trp andlac promoters. Among known eukaryotic promoters suitable in this regardare the CMV immediate early promoter, the HSV thymidine kinase promoter,the early and late SV40 promoters, the promoters of retroviral LTRs,such as those of the Rous sarcoma virus (“RSV”) and metallothioneinpromoters, such as the mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence.

Polypeptides can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., cited elsewhereherein.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG Also, generally, there will be a translation stopcodon at the end of the polypeptide and there will be a polyadenylationsignal and a transcription termination signal appropriately disposed atthe 3′ end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals. Thepolypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous fuctional regions Thus for instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification or during subsequent handling andstorage. Also, special regions also may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art For example, when large quantities of RG1 areneeded for the induction of antibodies, vectors which direct high levelexpression of fusion proteins that are readily purified may bedesirable. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the rg1 coding sequence may beligated into the vector in frame with sequence for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heede and Shuster, J. Biol. Chem.264-5503-5509, 1989) and the like. PTrcHis vectors (Invitrogen,Carlsbad, Calif.) may be used to express foreign polypeptides as fusionproteins containing a polyhistidine (6xHis) tag for rapid purification.Proteins made in such systems are designed to include cleavage sites,such as an enterokinase cleavage site, so that the cloned polypeptide ofinterest can be released from the fusion peptide moiety at will.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, inducible promoters, ifpresent, can be induced by appropriate means (e.g., temperature shift orexposure to chemical inducer) and cells cultured for an additionalperiod.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

The RG1 polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and or purification.Various other methods of protein purification well known in the artinclude those described in Deutscher, M., Methods in Enzymology, Vol182, Academic Press, San Diego, 1982; and Scopes, R., ProteinPurification: Principles and Practice Springer-Verlag, New York, 1982.

Alternatively, the polypeptides of the present invention can be producedby direct peptide synthesis using solid-phase techniques (Stewart etal., Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco,1969; Merrifield, J., J. Am. Chem. Soc. 85:2149-2154, 1963). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) in accordance with the instructions provided by themanufacturer. Various fragments of RG1 may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Uses of RG1 Polypeptides and the Polynucleotides which Encode Them

Rg1 polynucleotides and RG1 polypeptides may be used in accordance withthe present invention for a variety of applications, particularly thosethat make use of the chemical and biological properties of RG1.Additional applications relate to diagnosis and to treatment of diseasesof cell proliferation, such as prostate cancer. These aspects of theinvention are illustrated further by the following discussion and aredescribed further within the body of the specification.

The rationale for the use of the polynucleotide and polypeptidesequences of the present invention is based in part on the chemical andstructural homology between the RG1 disclosed herein and otherextracellular matrix molecules and on the preferential expression of RG1in prostate tissues as compared with other tissues. RG1 may be used inthe diagnosis and treatment of conditions, disorders or diseasesassociated with inappropriate growth of prostate tissue. These wouldinclude, but are not limited to, cancer and metastatic tumor growth.

Rg1 polynucleotide sequences can be used as DNA probes, and as targetsfor antisense and ribozyme therapy, or as templates for the productionof antisense polynucleotides.

RG1 polypeptides can be used to generate antibodies to RG1 that may beuseful in detecting the levels of RG1 polypeptide in cells and tissuesand in targeting drugs to primary and metastatic tumors.

RG1 polypeptides may be used to stimulate an immune response to RG1containing cells.

Polynucleotides encoding RG1 may be useful in diagnostic assays fordetecting the levels of polynucleotides encoding RG1 in cells andtissues.

In conditions associated with expression of RG1, such as prostatecancer, it may be advantageous to suppress expression or activity ofRG1. RG1 expression could be suppressed by administration of antisenseoligonucleotides or ribozymes. Alternatively, antibodies specificallyrecognizing areas of the RG1 polypeptide which are responsible for itsactivity may be administered to treat diseases or conditions associatedwith RG1 activity.

Polynucleotide Assays

This invention is also related to the use of the rg1-relatedpolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of rg1 polynucleotidesassociated with a disease state will provide a tool for the developmentof in vitro and in vivo diagnostics that can add or define a diagnosisof a disease or susceptibility to a disease that results from tissuespecific expression of RG1.

Individuals carrying mutations in the gene encoding RG1 may be detectedat the DNA level by a variety of techniques. Polynucleotide samples fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR prior to analysis (Saiki et al., Nature, 324 163-166, 1986).RNA or cDNA may also be used in the same ways. As an example, PCRprimers complementary to the polynucleotide sequence encoding RG1 can beused to identify and analyze rg1 expression and mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled rg1 RNAor alternatively, radiolabeled rg1 antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures Sequencedifferences between a reference gene and genes having mutations also maybe revealed by direct DNA sequencing. In addition, cloned DNA segmentsmay be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled polynucleotide orby automatic sequencing procedures with fluorescent tags

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242, 1985).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Catton et al., Proc. Natl. Acad. Sci., USA,85:4397-4401, 1985).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA).

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of RG1polypeptide in cells and tissues and body fluids, includingdetermination of normal and abnormal levels. Thus, for instance, adiagnostic assay in accordance with the invention for detectingover-expression of RG1 polypeptide compared to normal control tissuesamples may be used to detect the presence of neoplasia, for example,prostate cancer. Such diagnostic tests may be used to detect metastatictumor growth, as well. Assay techniques that can be used to determinelevels of a polypeptide, such as a RG1 polypeptide of the presentinvention, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays (RIA),competitive-binding assays, western Blot analysis and enzyme-linkedimmunoabsorbant assays (ELISA), and fluorescent activated cell sorting(FACS) Among these ELISAs frequently are preferred. An ELISA assayinitially comprises preparing an antibody specific to RG1, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as a radioactive, fluorescentor enzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the polypeptides inthe sample. Any free polypeptide binding sites on the dish are thencovered by incubating with a non-specific protein such as bovine serumalbumin. Next, the monoclonal antibody is incubated in the dish duringwhich time the monoclonal antibodies attach to any RG1 polypeptidesattached to the polystyrene dish. Unbound monoclonal antibody is washedout with buffer The reporter antibody linked to horseradish peroxidaseis placed in the dish resulting in binding of the reporter antibody toany monoclonal antibody bound to RG1. Unattached reporter antibody isthen washed out. Reagents for peroxidase activity, including acalorimetric substrate are then added to the dish. Immobilizedperoxidase, linked to RG1 through the primary and secondary antibodies,produces a colored reaction product. The amount of color developed in agiven time period indicates the amount of RG1 polypeptide present in thesample. Quantitative results typically are obtained by reference to astandard curve.

A competition assay may be employed wherein antibodies specific to RG1are attached to a solid support and labeled RG1 and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof RG1 in the sample.

These and other assays are described, among other places, in Hampton etal. (Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn., 1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

Antibodies

The invention further relates to antibodies that specifically bind toRG1, herein referred to as RG1 antibodies. The over-expression of RG1 inprostate tissues and its cell surface location represent characteristicsof an excellent marker for screening, diagnosis, prognosis, follow-upassays and imaging methods. In addition, these characteristics indicatethat RG1 may be an excellent target for therapeutic methods such astargeted antibody therapy, immunotherapy, and gene therapy. As usedherein, the term “specifically binds to” refers to the interaction of anantibody and a polypeptide, in which the interaction is dependent uponthe presence of a particular structure (i.e., the antigenic determinantor epitope) on the polypeptide; in other words, the antibody isrecognizing and binding to a specific polypeptide structure rather thanto proteins in general.

The RG1 polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto (Harlow, Antibodies, Cold Spring Harbor Press, NY(1989)). These antibodies can be, for example, polyclonal or monoclonalantibodies. The present invention also includes chimeric, single chain,humanized, and human antibodies, as well as Fab fragments, or theproduct of a Fab expression library. Various procedures known in the artmay be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature256: 495-497, 1975), the human B-cell hybridoma technique (Kozbor et al,Immunology Today 4: 72,1983) and the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al., in Monoclonal Antibodies andCancer, Alan R. Liss, Inc., 77-96, 1985).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al., Proc. Natl. Acad. Sci.USA 81:6851-6855, 1984; Neubergeretal., Nature 312:604-608,1984; Takedaet al., Nature 314:452-454, 1985). Alternatively, techniques describedfor the production of single chain antibodies (U.S. Pat. No. 4,946,778)can be adapted to produce RG1-specific single chain antibodies.

Furthermore, “human” antibodies can be produced using the methodsdescribed in U.S. Pat. Nos. 5,877,397 and 5,569,825, which areincorporated herein in full by reference.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Proc. Natl. Acad. Sci. USA 86:3833-3837, 1989) andWinter and Milstein (Nature 349:293-299, 1991).

Antibody fragments which contain specific binding sites for RG1 may alsobe generated. For example, such fragments include, but are not limitedto the F(ab′)2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science 256:1270-1281, 1989).

The amino acid sequence of RG1 presented herein may be used to selectspecific regions of the RG1 polypeptide for generating antibodies. Aswill be understood by those skilled in the art, the regions or epitopesof a RG1 polypeptide to which an antibody is directed may vary with theintended application. For example, antibodies intended for use in animmunoassay for the detection of membrane-bound RG1 on prostate cellsshould be directed toward accessible epitopes on the RG1 polypeptide.Regions of the RG1 polypeptide that show immunogenic structure, as wellas other regions and domains, can readily be identified using variousother methods known in the art, such as Chou-Fasman, Garnier-Robson, orJameson-Wolf analysis. Fragments containing these residues areparticularly suited in generating anti-RG1 antibodies. Particularlyuseful fragments include, but are not limited to, the sequencesPLGGESICSAGAPAKYSIT (SEQ ID NO: 8); HSSDYSMWRKNQYVS (SEQ ID NO: 10);DAGTDSGFTFSSPNFATIPQDTV (SEQ ID NO: 11); and NEIVDSASVPET (SEQ ID NO:12). Generation of polyclonal antibodies to these regions is describedin Example 4.

RG1 antibodies of the invention may be particularly useful in diagnosticassays, imaging methodologies, and therapeutic methods for themanagement of prostate cancer. The invention provides variousimmunological assays useful for the detection of RG1 polypeptides andfor the diagnosis of prostate cancer. Such assays generally comprise oneor more RG1 antibodies capable of recognizing and binding a RG1polypeptide. The most preferred antibodies will selectively bind to RG1and will not bind (or bind weakly) to non-RG1 polypeptides. The assaysinclude various immunological assay formats well known in the art,including but not limited to various types of radioimmunoassays,enzyme-linked immunoabsorbent assays, and the like. In addition,immunological Imaging methods capable of detecting prostate cancer arealso provided by the invention, including but not limited toradioscintigraphic imaging methods using labeled RG1 antibodies. Suchassays may be clinically useful in the detection, monitoring andprognosis of prostate cancer.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Additionally, RG1 antibodies may be used to isolate RG1 positive cellsusing cell sorting and purification techniques. In particular, RG1antibodies may be used to isolate prostate cancer cells from xenografttumor tissue, from cells in culture, etc. using antibody-based cellsorting or affinity purification techniques. Other uses of the RG1antibodies of the invention include generating anti-idiotypic antibodiesthat mimic the RG1 polypeptide.

The RG1 antibodies can be used for detecting the presence of prostatecancer or tumor metastasis. The presence of such RG1-containing cellswithin various biological samples, including serum, prostate and othertissue biopsy specimens, may be detected with RG1 antibodies. Inaddition, RG1 antibodies may be used in various imaging methodologiessuch as immunoscintigraphy with Tc-99m (or other isotope) conjugatedantibody. For example, an Imaging protocol similar to the one recentlydescribed using an In-111 conjugated anti-PSMA antibody may be used todetect recurrent and metastatic prostate carcinomas (Sodee et al., Clin.Nuc. Med. 21: 759-766, 1997).

The RG1 antibodies of the invention may be labeled with a detectablemarker or conjugated to a second molecule, such as a cytotoxic agent,and used for targeting the second molecule to a RG1 positive cell(Vitetta, E. S. et al., Immunotoxin Therapy, in DeVita, Jr, V. T. etal., eds, Cancer: Principles and Practice of Oncology, 4^(th) ed., J.B.Lippincott Co., Philadelphia, 2624-2636, 1993). Examples of cytotoxicagents include, but are not limited to ricin, doxorubicin, daunorubicin,taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diptheria toxin, Pseudomonas exotoxin(PE) A, PE40, abrin, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes. Suitable detectable markers include, but are not limitedto, a radioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. Suitableradioisotopes include the following: Antimony-124, Antimony-125,Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-j206,Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139,Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-56, Cobalt-57,Cobalt-58, Cobalt-60, Cobalt-64, Erbium-169, Europium-152,Gadolinium-153, Gold-195, Gold-199, Hafnium-175, Hafnium-181, Indium-11,Iodine-123, Iodine-131, Iridium-192, Iron-55, Iron-59, Krypton-85,Lead-210, Manganese-54, Mercury-197, Mercury-203, Molybdenum-99,Neodymium-147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191,Palladium-103, Platinum-195m, Praseodymium-143, Promethium-147,Protactinium-233, Radium-2226, Rhenium-186, Rubidium-86, Ruthenium-103,Ruthenium-106, Scandium-44, Scandium-46, Selenium-75, Silver-110m,Silver-11, Sodium-22, Strontium-85, Strontium-89, Strontium-90,Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125, Tellurium-132,Thallium-170, Thallium-204, Thorium-228, Thorium-232, Tin-113,Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169,Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, and Zirconium-95.

Immunotherapy for Prostate Cancer

The invention provides various immunotherapeutic methods for treatingprostate cancer, including antibody therapy, in vivo vaccines, and exvivo immunotherapy approaches. In one approach, the invention providesRG1 antibodies which may be used systemically to treat prostate cancer.For example, unconjugated RG1 antibodies may be introduced into apatient such that the antibody binds to RG1 on, in or associated withprostate cancer cells and mediates the destruction of the cells, and thetumor, by mechanisms which may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, altering the physiologicfunction of RG1, and/or the inhibition of ligand binding or signaltransduction pathways. RG1 antibodies conjugated to toxic agents such asricin or radioisotopes may also be used therapeutically to deliver thetoxic agent directly to RG1-bearing prostate tumor cells and therebydestroy the tumor cells.

Prostate cancer immunotherapy using RG1 antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., Crit. Rev. Immunol. 18: 133-138,1998), multiple myeloma (Ozaki et al., Blood 90: 3179-3186, 1997;Tsunenari et al., Blood 90: 2437-2444, 1997), gastric cancer (Kasprzyket al, Cancer Res. 52: 2771-2776, 1992), B-cell lymphoma (Funakoshi etal., Immunther. Emphasis Tumor Immunol. 19: 93-101, 1996), leukemia(Zhong et al., Leuk. Res. 20: 581-589, 1996), colorectal cancer (Moun etal., Cancer Res. 54 6160-6166, 1994; Velders et al., Cancer Res.55-4398-4403, 1995), and breast cancer (Shepard et al., J. Clin.Immunol. 11: 117-127, 1991).

The invention further provides vaccines formulated to contain a RG1polypeptide or fragment thereof. The use of a tumor antigen in a vaccinefor generating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., Int. J.Cancer 63: 231-237, 1995; Fong et al., J. Immunol. 159: 3113-3117,1997). Such methods can be readily practiced by employing a RG1polypeptide, or fragment thereof, or a RG1-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the RG1 immunogen.

For example, viral gene delivery systems may be used to deliver aRG1-encoding nucleic acid molecule. Various viral gene delivery systemswhich can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowipox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, in Curr. Opin, Immunol. 8: 658-663, 1996).Non-viral delivery systems may also be employed by using naked DNAencoding a RG1 polypeptide or fragment thereof introduced into thepatient (i.e., intramuscularly) to induce an anti-tumor response In oneembodiment, the full-length human rg1 cDNA may be employed. In anotherembodiment, human rg1 cDNA fragments may be employed. In anotherembodiment, rg1 nucleic acid molecules encoding specific T lymphocyte(CTL) epitopes may be employed. CTL epitopes can be determined usingspecific algorithims (e.g., Epimer, Brown University) to identifypeptides within a RG1 polypeptide which are capable of optimally bindingto specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present a RG1 polypeptide as antigen to apatient's immune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., Prostate 28: 65-69, 1996; Murphy et al.,Prostate 29: 371-380, 1996). Dendritic cells can be used to present RG1polypeptides to T cells in the context of MHC class I and II molecules.In one embodiment, autologous dendritic cells are pulsed with RG1polypeptides capable of binding to MHC molecules. In another embodiment,dendritic cells are pulsed with the complete RG1 polypeptide. Yetanother embodiment involves engineering the overexpression of the rg1gene in dendritic cells using various implementing vectors known in theart, such as adenovirus (Arthur et al., Cancer Gene Ther. 4:17-25,1997),retrovirus (Henderson et al., Cancer Res. 56: 3763-3770,1996),lentivirus, adeno-associated virus, DNA transfection (Ribas et al.,Cancer Res. 57: 2865-2869, 1997), and tumor-derived RNA transfection(Ashley et al., J. Exp. Med. 186: 1177-1182, 1997).

Anti-idiotypic anti-RG1 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga RG1 polypeptide. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can be readily adapted togenerate anti-idiotypic anti-RG1 antibodies that mimic an epitope on aRG1 polypeptide (see, for example, Wagner et al., Hybridoma 16: 33-40,1997: Foon et al., J. Clin. Invest. 96: 334-342, 1995; Herlyn et al.,Cancer Immunol Immunother 43: 65-76, 1996) Such an anti-idiotypicantibody can be used in anti-idiotypic therapy as presently practicedwith other anti-idiotypic antibodies directed against tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing RG1. Using the RG1-encoding DNA moleculesdescribed herein, constructs comprising DNA encoding a RG1polypeptide/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take up the construct and express theencoded RG1 polypeptide/immunogen. The RG1 polypeptide/immunogen may beexpressed as a cell surface polypeptide or be secreted. Expression ofthe RG1 polypeptide/immunogen results in the generation of prophylacticor therapeutic humoral and cellular immunity against prostate cancer.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used (for a review, see information andreferences published at internet address www.genweb.com).

Anti-sense Oligonucleotides, Antisense Vectors, and Ribozymes

Anti-sense polynucleotides complementary to rg1 may be preparedsynthetically. Such oligonucleotides may be delivered into cells with orwithout lipids that may assist uptake of the anti-sense oligonucleotidesinto cells.

Alternatively, expression vectors derived from retroviruses, adenovirus,herpes or vaccinia viruses, or from various bacterial plasmids, may alsobe used for construction and delivery of recombinant vectors which willexpress anti-sense rg1. See, for example, the techniques described inSambrook et al. (supra) and Ausubel et al. (supra).

The polynucleotides comprising the full length cDNA sequence and/or itsregulatory elements enable researchers to use rg1 polynucleotides as aninvestigative tool in sense strands (Youssoufian and Lodish, Mol. Cell.Biol. 13:98-104,1993) or antisense strands (Eguchi, et al., Annu. Rev.Biochem. 60:631-652, 1991) for the regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligomers, or larger fragments, can be designed from various locationsalong the coding or control regions.

Genes encoding RG1 can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a desired rg1polynucleotide fragment. Such constructs can flood cells withuntranslatable sense or antisense sequences. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until all copies are disabled by endogenous nucleases.Transient expression may last for a month or more with a non-replicatingvector and even longer if appropriate replication elements are part ofthe vector system.

As mentioned above, modification of gene expression can be obtained bydesigning antisense molecules, DNA or RNA, to control regions of rg1,i.e., the promoters, enhancers, and introns. Oligonucleotides derivedfrom the transcription initiation site, e.g. between −10 and +10 regionsof the leader sequence, are preferred. The antisense molecules may alsobe designed to block translation of mRNA by preventing the transcriptfrom binding to ribosomes. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology. Triple helix pairingcompromises the ability of the double helix to open sufficiently for thebinding of polymerases, transcription factors, or regulatory molecules.Recent therapeutic advances using triplex DNA were reviewed by Gee, J.E. et al. (In Huber and Car, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., 1994).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA (U.S. Pat. No. 4,987,071; WO 93/23057). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Within the scope of the invention are engineered hammerheadmotif ribozyme molecules that can specifically and efficiently catalyzeendonucleolytic cleavage of RNA encoding RG1. Specific ribozyme cleavagesites within any potential RNA target are initially identified byscanning the target molecule for ribozyme cleavage sites which includethe following sequences, GUA, GUU and GUC. Once identified, short RNAsequences of between 15 and 20 ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor secondary structural features which may render the oligonucleotideinoperable. The suitability of candidate targets may also be evaluatedby testing accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays (Irie et al.,Advance. Pharmacol. 40:207-257, 1997).

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription or byDNA sequences encoding RG1. Such DNA sequences may be incorporated intoa wide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculesor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule.Increased stability can also be achieved by the inclusion ofnontraditional bases such as inosine and queosine as well as acetyl-,methyl-, thio- and similarly modified forms of adenine, cytidine,guanine, thymine, and uridine which are not as easily recognized byendogenous endonucleases.

Methods for introducing antisense vectors into cells or tissues includethose methods discussed infra and which are equally suitable for invivo, in vitro and ex vivo therapy. For ex vivo therapy, antisensevectors are introduced into cells taken from the patient and clonallypropagated for autologous transplant back into that same patient aspresented in U.S. Pat. Nos. 5,399,493 and 5,437,994, disclosed herein byreference. Delivery by transfection and by liposome or other lipid basedor non-lipid based agents are well known in the art.

Assays for Identifying Agents Binding to RG1

The present invention also relates to assays and methods which can beused to identify agents that bind to RG1. Specifically, agents that bindto RG1 can be identified by the ability of the RG1 ligand or other agentor constituent to bind to RG1 and/or the ability to inhibit/stimulateRG1 activity.

Alternatively, agents that bind to a RG1 polypeptide can be identifiedusing a yeast two-hybrid system or a binding capture assay. In the yeasttwo hybrid system, an expression unit encoding a fusion protein made upof one subunit of a two subunit transcription factor and the RG1polypeptide is introduced and expressed in a yeast cell. The cell isfurther modified to contain (1) an expression unit encoding a detectablemarker whose expression requires the two subunit transcription factorfor expression and (2) an expression unit that encodes a fusion proteinmade up of the second subunit of the transcription factor and a clonedsegment of DNA. If the cloned segment of DNA encodes a protein thatbinds to the RG1 polypeptide, the expression results in the interactionof RG1 and the encoded protein. This brings the two subunits of thetranscription factor into binding proximity, allowing reconstitution ofthe transcription factor. This results in expression of the detectablemarker. The yeast two hybrid system is particularly useful in screeninga library of cDNA encoding segments for cellular binding partners ofRG1.

RG1 polypeptides which may be used in the above assays include, but arenot limited to, an isolated RG1 polypeptide, a fragment of a RG1polypeptide, a cell that has been altered to express a RG1 polypeptide,or a fraction of a cell that has been altered to express a RG1polypeptide. Further, the RG1 polypeptide can be the entire polypeptideor a defined fragment of the RG1 polypeptide. It will be apparent to oneof ordinary skill in the art that so long as the RG1 polypeptide can beassayed for agent binding, e.g. by a shift in molecular weight oractivity, the present assay can be used.

The method used to identify whether an agent/cellular component binds toa RG1 polypeptide will be based primarily on the nature of the RG1polypeptide used. For example, a gel retardation assay can be used todetermine whether an agent binds to RG1 or a fragment thereof.Alternatively, immunodetection and biochip technologies can be adoptedfor use with the RG1 polypeptide. A skilled artisan can readily employnumerous art-known techniques for determining whether a particular agentbinds to an RG1 polypeptide.

Agents and cellular components can be further tested for the ability tomodulate the activity of an RG1 polypeptide using a cell-free assaysystem or a cellular assay system. As the activities of the RG1polypeptide become more defined, functional assays based on theidentified activity can be employed.

As used herein, an agent is said to antagonize RG1 activity when theagent reduces RG1 activity. The preferred antagonist will selectivelyantagonize RG1, not affecting any other cellular proteins. Further, thepreferred antagonist will reduce RG1 activity by more than 50%, morepreferably by more than 90%, most preferably eliminating all RG1activity.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences of the RG1 polypeptide. An example of randomlyselected agents is the use of a chemical library or a peptidecombinatorial library, or growth broth of an organism or plant extract.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis that takes into accountthe sequence of the target site an/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that make up the RG1polypeptide. For example, a rationally selected peptide agent can be apeptide whose amino acid sequence is identical to a fragment of an RG1polypeptide.

The agents tested in the methods of the present invention can be, asexamples, peptides, antibodies, oligonucleotides, small molecules andvitamin derivatives, as well as carbohydrates. A skilled artisan canreadily recognize that there is no limit as to the structural nature ofthe agents used in the present screening method. One class of agents ofthe present invention are peptide agents whose amino acid sequences arechosen based on the amino acid sequence of the RG1 polypeptide.

Peptide agents can be prepared using standard solid phase (or solutionphase) peptide synthesis methods, as is known in the art. In addition,the DNA encoding these peptides may be synthesized using commerciallyavailable oligonucleotide synthesis instrumentation and producedrecombinantly using standard recombinant production systems. Theproduction using solid phase peptide synthesis is necessitated ifno-gene-encoded amino acids are to be included.

Another class of agent of the present invention are antibodiesimmunoreactive with critical positions of the RG1 polypeptide. Asdescribed above, antibodies are obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions, thoseportions of the RG1 polypeptide intended to be targeted by theantibodies. Such agents can be used in competitive binding studies toidentify second generation inhibitory agents as well as to block RG1activity.

The cellular extracts tested in the methods of the present invention canbe, as examples, aqueous extracts of cells or tissues, organic extractsof cells or tissues or partially purified cellular fractions A skilledartisan can readily recognize that there is no limit as to the source ofthe cellular extract used in the screening method of the presentinvention.

Agents that bind a RG1 polypeptide, such as a RG1 antibody, can be usedto modulate the activity of RG1,to target anticancer agents toappropriate mammalian cells, or to identify agents that block theinteraction with RG1. Cells expressing RG1 can be targeted or identifiedby using an agent that binds to RG1.

How the RG1 binding agents will be used depends on the nature of the RG1binding agent. For example, a RG1 binding agent can be used to: deliverconjugated toxins, such as diphtheria toxin, cholera toxin, ricin orpseudomonas exotoxin, to a RG1 expressing cell; modulate RG1 activity;to directly kill a RG1 expressing cell; or in screens to identifycompetitive binding agents. For example, a RG1 inhibitory agent can beused to directly inhibit the growth of RG1 expressing cells whereas aRG1 binding agent can be used as a diagnostic agent.

Pharmaceutical Compositions and Administration

The present invention also relates to pharmaceutical compositions whichmay comprise rg1 polynucleotides, RG1 polypeptides, antibodies,agonists, antagonists, or inhibitors, alone or in combination with atleast one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Any of these molecules can be administered to a patient alone, orin combination with other agents, drugs or hormones, in pharmaceuticalcompositions where it is mixed with excipient(s) or pharmaceuticallyacceptable carriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert.

The present invention also relates to the administration ofpharmaceutical compositions. Such administration is accomplished orallyor parenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxilliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co,Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxilliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl, cellulose, hydroxypropylmethylcellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, andoptionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances that increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Manufacture and Storage.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with may acids, including by not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of RG1, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose, i.e. treatment of aparticular disease state characterized by RG1 expression. Thedetermination of an effective dose is well within the capability ofthose skilled in the art.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors which ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations what include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations forpolynucleotides than for proteins or their inhibitors. Similarly,delivery of polynucleotides or polypeptides will be specific toparticular cells, conditions, locations, etc.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

EXAMPLE 1 Identification of Human rg1 Polynucleotide

Rg1 was identified as a gene expressed in the prostate by miningIncyte's LifeSeq database. The nucleotide sequence was identified by anannotation serch of the database, using the “Protein Function” toolprovided by Incyte for the purpose of searching the database. Thenucleotide sequence was found in the category of cell adhesion moleculesin the annotated database and was described as a homologue of f-spondin.Electronic Northern analysis of the distribution of rg1 polynucleotidesequences in the set of libraries in the database revealed that rg1 wasexpressed at high levels in the prostate libraries and at lower levelsin a number of other tissue libraries, including those from normal andtumor tissues.

Following assembly of the set of rg1 clones in the database into acontiguous polynucleotide sequence, and editing of the contiguoussequence, a full-length coding sequence was identified in the predictedassembled polynucleotide. This sequence coded for a protein homologousto f-spondin and to Mindin-2.

Incyte clones 1640796, 1712252, and 1880265 were obtained from Incytefor experimental work and clone 3360733 was identified as containing themost 5′ nucleotide sequence. This clone was fully sequenced andcontained the full coding sequence for the predicted RG1 protein. Thissequence is shown in FIG. 1 (SEQ D NO: 1).

EXAMPLE 2 Rg1 mRNA Expression

The expression of rg1 mRNA in a variety of samples from normal and tumortissues and in cell lines, was determined by semi-quantitative PCR usinga Taqman assay, (Perkin-Elmer). Prostate normal, benign and tumor tissuesamples that had been graded according to a modified Gleason gradingsystem were obtained from the Urology Department at Stanford UniversitySchool of Medicine. RNA was isolated from these by standard procedures.RNA from other tumor and normal tissues was purchased from commercialsources, including Clonetech, and Biochain. Prostate tumor cell lines,(PC-3, LNCaP and DU145), were obtained from American Type CultureCollection and propagated in culture by standard methods using serumcontaining medium. Xenograft tumors derived from these cell lines wereestablished in nude mice and harvested from the mice approximately 4-6weeks after implantation. RNA was isolated from the tumors by standardprocedures.

Taqman based PCR analysis was performed using the primers CGC GCA TAGCTC CGA CTA C (SEQ ID NO: 3) and GCC GCG TCC GCA AAG (SEQ ID NO: 4) andthe Taqman probe: 6-FAM-AGG AAG AAC CAG TAC GTC AGT AAC GGG CTG-Tamra(SEQ ID NO: 5).

These primers and probe were designed using Perkin Elmer's PrimerExpress software and were synthesized by Synthetic Genetics. PCRreactions were carried out for 30-40 cycles and quantified usingprostate RNA to generate a standard curve for relative comparison. Thisanalysis demonstrated that rg1 mRNA was detected at highest abundance inthe prostate and at significantly lower levels in several other tissues(See FIG. 5).

EXAMPLE 3 Cloning and Expression of RG1 in BHK Cells

The RG1 coding region was obtained from Incyte plasmid 3360733. Thecoding sequence was PCR amplified with primers SST115(5′-TCCCTCTAGAGCCACCATGGAAAACCCCAGCCCGGC -3′) (SEQ ID NO: 6) and SST113(5′-AAGGCATCACGTGTTAGACGCAGTTATCAGGGACG-3′) (SEQ ID NO: 7) in a standardPCR reaction (100 ul) using 1×Pfu Turbo polymerase buffer (Stratagene,La Jolla, Calif.)/200 uM dNTPs/0.2 uM oligonucleotide primers/2.5 U PfuTurbo polymerase (Stratagene). PCR amplification conditions were asfollows: 3 mins at 95° C., (15 seconds at 95° C., 30 seconds at 60° C.,2 minutes at 72° C.)×35, 72° C. for 7 minutes. The resulting PCRamplified product was purified using a QlAquick PCR column (Qiagen,Valencia, Calif.) and digested with Xbal and PmII restriction enzymes toresult in a 1010 bp fragment that was purified from a 1% agarose gelusing a BIO 101 GeneClean Kit (Vista, Calif.). The purified fragment wasligated (using Epicientre Fast Link Kit, (Epicenter, Madison, Wis.) tothe noncytopathic Sindbis expression vector pSINrep21(Agapov et al,1998, PNAS 95: 12989-12994) digested with XbaI and PmII, and transformedinto DH5 alpha competent cells (Life Technologies, Gaithersburg, Calif.)and selected on LB agar plates containing ampicillin (100 ug/ml). Onesuch ampicillin resistant colony was grown in LB medium with ampicillinand shown by sequence analysis to contain the inserted RG1 codingsequence. This plasmid was called pPEG6.

Two micrograms of pPEG6 was used to transfect 1-3×10⁵ bovine hamsterkidney cells (BHK) cells using Lipofectamine Plus reagent (LifeTechnologies, Gaithersburg, Md.) according to the manufacturer'sinstructions. Following transfection, cells were incubated in DMEM plusfetal blood serum for 24-48 hours, at which time the cells were split 1to 10 and selection for the plasmid containing cells was initiated byadding puromycin (2.5 ug/ml final concentration) and DMEM containingserum. After the cells were confluent (4-5 days post puromycin addition)the cells were washed with PBS, split 1 to 10, and DMEM medium withserum and 5 ug/ml puromycin was added. After an additional 2-3 days, themedium was replaced with DMEM and 5 ug/ml puromycin without serum, grownfor 2-3 days and the presence of RG1 protein was detected in the mediumby Western analysis using RG1 antibodies. RG1 protein was detected at alevel of 1 ug/ml.

EXAMPLE 4 Antibody Generation

Rabbit polyclonal antisera were raised against five syntheticpolypeptide sequences derived from the RG1 protein sequence. Thesesequences were selected because of their predicted positions at thesurface of the protein, in order to generate antisera that are morelikely to recognize surface epitopes. Cysteine residues were replacedwith aminobutyric acid (Abu) to aid synthesis. The specific amino acidsequences, positions on the RG1 protein and designations for the fivepeptides are listed below.

Designation Position Amino Acid Sequence 1C 28-46 PLGGESICSAGAPAKYSIT(SEQ ID NO: 8) 2C 46-64 TFTGKWSQTAFPKQYPLFR (SEQ ID NO: 9) 3C 77-91HSSDYSMWRKNQYVS (SEQ ID NO: 10) 4C 188-210 DAGTDSGFTFSSPNFATIPQDTV (SEQID NO: 11) 5C 263-274 NEIVDSASVPET (SEQ ID NO: 12)

Peptides were covalently coupled to keyhole limpet hemocyanin (KLH), viaan additional carboxyl-terminal cysteine, for use as an immunogen.Similarly, a bovine serum albumin (BSA) conjugate was prepared for theanalysis of antisera titers via ELISA.

Two animals were immunized with each peptide. Initial immunizations wereperformed in Freunds complete adjuvant (0.5 mg/animal), followed byboosts at three week intervals with 0.25 mg/animal in Freunds incompleteadjuvant applied intramuscularly. Periodic test bleeds were taken andantibody titers against the specific BSA-peptide conjugate were measuredby ELISA and compared with preimmune sera Antisera against peptides 1Cand 3C were shown to be active. Antisera against peptide 2C did notrecognize RG1 polypeptide. Antisera against peptides 4C and 5C were nottested.

Human monoclonal antibodies against RG1 were generated by immunizingtransgenic mice against RG1 peptides and a 6-histidine-tagged RG1 fusionprotein expressed in E. coli. Splenocytes of these animals were fusedwith myeloma cells to produce hybridoma cells. The resulting hybridomaswere screened by ELISA for those producing antibodies directed againstRG1 peptides and protein.

EXAMPLE 5 Western Blot Analysis of Antibodies

Antisera were tested for RG1 specificity via Western blotting. RG1specific antisera (those raised against sequences 1C and 3C, above) weretested on RG1 transiently expressed in COS cells, native RG1 secretedfrom LNCaP cells and RG1 produced from transfected baby hamster kidneycells (BHK). RG1-specific antisera were further tested on lysatesprepared from: LNCaP tumors, LNCaP cells, PC3 tumors, PC3 cells andseveral clinical samples of human prostate tumors. Cells and tissueswere lysed in detergent buffer. After boiling for 5 min, 10 ul of eachlysate was loaded onto a 12% SDS-polyacrylamide gel to resolve proteins.Separated proteins were then transferred to nitrocellulose membranes.Binding specificity of RG1 antibodies was verified by binding in thepresence of the homologous and heterologous peptides. RG1-specificantisera could detect the protein in all samples but PC-3 cells and PC-3tumors.

EXAMPLE 6 Purification of Native RG1 Protein Secreted from LNCaP Cells

LNCaP cells grown in culture were shown to secrete native RG1 protein byWestern blot analysis. In order to purify the native protein, cells weregrown for 48 hours in media lacking serum This serum-free conditionedmedia was harvested, centrifuged to remove any cells, and concentratedapproximately fifty-fold by ultrafiltration. The concentrated media wasthen diluted ten-fold with 20 mM sodium acetate buffer, pH 6.5 andloaded onto a Q-Sepharose anion exchange column. Column elutionconsisted of a sodium chloride gradient (0.5% per minute) whilecollecting 2.0 ml fractions. The RG1 protein eluted at approximately 75mM NaCl as determined by Western blot and SDS PAGE. The native RG1protein runs at a slightly lower molecular weight than the 6histidine-RG1 fusion protein expressed in bacteria, presumably becauseit lacks the fusion peptide.

EXAMPLE 7 Immunohistochemical Staining of RG1 Expression

The expression of RG1 protein was determined by LifeSpan Biosciences,Inc. in a variety of human tissues, including kidney, lung, pancreas,muscle, brain and prostate. Additional prostate tissues were obtainedfrom the Urology Department at Stanford University School of Stanfordand tested at Berlex The tissue sections were deparaffinized usingstandard procedures. The polyclonal antibody RG1-3C was used as aprimary antibody and the detection system consisted of using VectorABC-AP kit (AK5002) with a Vector red substrate kit (Sk5002). As anegative control, the staining was carried out in the absence of theprimary antibody.

All publications and patents mentioned in the above specification areherein incorporated by reference. While the present invention has beendescribed with reference to the specific embodiments thereof, it shouldbe understood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from the truespirit and scope of the invention. In addition, many modifications maybe made to adapt a particular situation, material, composition ofmatter, process, process step or steps, to the objective, spirit andscope of the present invention. All such modifications are intended tobe within the scope of the claims appended hereto

13 1 1785 DNA Homo sapiens CDS (296)..(1291) 1 agaaaggggt gcggcagcactgccagggga agagggtgat ccgacccggg gaaggtcgct 60 gggcagggcg agttgggaaagcggcagccc ccgccgcccc cgcagcccct tctcctcctt 120 tctcccacgt cctatctgcctctcgctgga ggccaggccg tgcagcatcg aagacaggag 180 gaactggagc ctcattggccggcccggggc gccggcctcg ggcttaaata ggagctccgg 240 gctctggctg ggacccgaccgctgccggcc gcgctcccgc tgctcctgcc gggtg atg 298 Met 1 gaa aac ccc agc ccggcc gcc gcc ctg ggc aag gcc ctc tgc gct ctc 346 Glu Asn Pro Ser Pro AlaAla Ala Leu Gly Lys Ala Leu Cys Ala Leu 5 10 15 ctc ctg gcc act ctc ggcgcc gcc ggc cag cct ctt ggg gga gag tcc 394 Leu Leu Ala Thr Leu Gly AlaAla Gly Gln Pro Leu Gly Gly Glu Ser 20 25 30 atc tgt tcc gcc gga gcc ccggcc aaa tac agc atc acc ttc acg ggc 442 Ile Cys Ser Ala Gly Ala Pro AlaLys Tyr Ser Ile Thr Phe Thr Gly 35 40 45 aag tgg agc cag acg gcc ttc cccaag cag tac ccc ctg ttc cgc ccc 490 Lys Trp Ser Gln Thr Ala Phe Pro LysGln Tyr Pro Leu Phe Arg Pro 50 55 60 65 cct gcg cag tgg tct tcg ctg ctgggg gcc gcg cat agc tcc gac tac 538 Pro Ala Gln Trp Ser Ser Leu Leu GlyAla Ala His Ser Ser Asp Tyr 70 75 80 agc atg tgg agg aag aac cag tac gtcagt aac ggg ctg cgc gac ttt 586 Ser Met Trp Arg Lys Asn Gln Tyr Val SerAsn Gly Leu Arg Asp Phe 85 90 95 gcg gag cgc ggc gag gcc tgg gcg ctg atgaag gag atc gag gcg gcg 634 Ala Glu Arg Gly Glu Ala Trp Ala Leu Met LysGlu Ile Glu Ala Ala 100 105 110 ggg gag gcg ctg cag agc gtg cac gcg gtgttt tcg gcg ccc gcc gtc 682 Gly Glu Ala Leu Gln Ser Val His Ala Val PheSer Ala Pro Ala Val 115 120 125 ccc agc ggc acc ggg cag acg tcg gcg gagctg gag gtg cag cgc agg 730 Pro Ser Gly Thr Gly Gln Thr Ser Ala Glu LeuGlu Val Gln Arg Arg 130 135 140 145 cac tcg ctg gtc tcg ttt gtg gtg cgcatc gtg ccc agc ccc gac tgg 778 His Ser Leu Val Ser Phe Val Val Arg IleVal Pro Ser Pro Asp Trp 150 155 160 ttc gtg ggc gtg gac agc ctg gac ctgtgc gac ggg gac cgt tgg cgg 826 Phe Val Gly Val Asp Ser Leu Asp Leu CysAsp Gly Asp Arg Trp Arg 165 170 175 gaa cag gcg gcg ctg gac ctg tac ccctac gac gcc ggg acg gac agc 874 Glu Gln Ala Ala Leu Asp Leu Tyr Pro TyrAsp Ala Gly Thr Asp Ser 180 185 190 ggc ttc acc ttc tcc tcc ccc aac ttcgcc acc atc ccg cag gac acg 922 Gly Phe Thr Phe Ser Ser Pro Asn Phe AlaThr Ile Pro Gln Asp Thr 195 200 205 gtg acc gag ata acg tcc tcc tct cccagc cac ccg gcc aac tcc ttc 970 Val Thr Glu Ile Thr Ser Ser Ser Pro SerHis Pro Ala Asn Ser Phe 210 215 220 225 tac tac cca cgg ctg aag gcc ctgcct ccc atc gcc agg gtg aca ctg 1018 Tyr Tyr Pro Arg Leu Lys Ala Leu ProPro Ile Ala Arg Val Thr Leu 230 235 240 gtg cgg ctg cga cag agc ccc agggcc ttc atc cct ccc gcc cca gtc 1066 Val Arg Leu Arg Gln Ser Pro Arg AlaPhe Ile Pro Pro Ala Pro Val 245 250 255 ctg ccc agc agg gac aat gag attgta gac agc gcc tca gtt cca gaa 1114 Leu Pro Ser Arg Asp Asn Glu Ile ValAsp Ser Ala Ser Val Pro Glu 260 265 270 acg ccg ctg gac tgc gag gtc tccctg tgg tcg tcc tgg gga ctg tgc 1162 Thr Pro Leu Asp Cys Glu Val Ser LeuTrp Ser Ser Trp Gly Leu Cys 275 280 285 gga ggc cac tgt ggg agg ctc gggacc aag agc agg act cgc tac gtc 1210 Gly Gly His Cys Gly Arg Leu Gly ThrLys Ser Arg Thr Arg Tyr Val 290 295 300 305 cgg gtc cag ccc gcc aac aacggg agc ccc tgc ccc gag ctc gaa gaa 1258 Arg Val Gln Pro Ala Asn Asn GlySer Pro Cys Pro Glu Leu Glu Glu 310 315 320 gag gct gag tgc gtc cct gataac tgc gtc taa gaccagagcc ccgcagcccc 1311 Glu Ala Glu Cys Val Pro AspAsn Cys Val 325 330 tggggccccc cggagccatg gggtgtcggg ggctcctgtgcaggctcatg ctgcaggcgg 1371 ccgagggcac agggggtttc gcgctgctcc tgaccgcggtgaggccgcgc cgaccatctc 1431 tgcactgaag ggccctctgg tggccggcac gggcattgggaaacagcctc ctcctttccc 1491 aaccttgctt cttaggggcc cccgtgtccc gtctgctctcagcctcctcc tcctgcagga 1551 taaagtcatc cccaaggctc cagctactct aaattatgtctccttataag ttattgctgc 1611 tccaggagat tgtccttcat cgtccagggg cctggctcccacgtggttgc agatacctca 1671 gacctggtgc tctaggctgt gctgagccca ctctcccgagggcgcatcca agcgggggcc 1731 acttgagaag tgaataaatg gggcggtttc ggaagcgtcaaaaaaaaaaa aaaa 1785 2 331 PRT Homo sapiens 2 Met Glu Asn Pro Ser ProAla Ala Ala Leu Gly Lys Ala Leu Cys Ala 1 5 10 15 Leu Leu Leu Ala ThrLeu Gly Ala Ala Gly Gln Pro Leu Gly Gly Glu 20 25 30 Ser Ile Cys Ser AlaGly Ala Pro Ala Lys Tyr Ser Ile Thr Phe Thr 35 40 45 Gly Lys Trp Ser GlnThr Ala Phe Pro Lys Gln Tyr Pro Leu Phe Arg 50 55 60 Pro Pro Ala Gln TrpSer Ser Leu Leu Gly Ala Ala His Ser Ser Asp 65 70 75 80 Tyr Ser Met TrpArg Lys Asn Gln Tyr Val Ser Asn Gly Leu Arg Asp 85 90 95 Phe Ala Glu ArgGly Glu Ala Trp Ala Leu Met Lys Glu Ile Glu Ala 100 105 110 Ala Gly GluAla Leu Gln Ser Val His Ala Val Phe Ser Ala Pro Ala 115 120 125 Val ProSer Gly Thr Gly Gln Thr Ser Ala Glu Leu Glu Val Gln Arg 130 135 140 ArgHis Ser Leu Val Ser Phe Val Val Arg Ile Val Pro Ser Pro Asp 145 150 155160 Trp Phe Val Gly Val Asp Ser Leu Asp Leu Cys Asp Gly Asp Arg Trp 165170 175 Arg Glu Gln Ala Ala Leu Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp180 185 190 Ser Gly Phe Thr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro GlnAsp 195 200 205 Thr Val Thr Glu Ile Thr Ser Ser Ser Pro Ser His Pro AlaAsn Ser 210 215 220 Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile AlaArg Val Thr 225 230 235 240 Leu Val Arg Leu Arg Gln Ser Pro Arg Ala PheIle Pro Pro Ala Pro 245 250 255 Val Leu Pro Ser Arg Asp Asn Glu Ile ValAsp Ser Ala Ser Val Pro 260 265 270 Glu Thr Pro Leu Asp Cys Glu Val SerLeu Trp Ser Ser Trp Gly Leu 275 280 285 Cys Gly Gly His Cys Gly Arg LeuGly Thr Lys Ser Arg Thr Arg Tyr 290 295 300 Val Arg Val Gln Pro Ala AsnAsn Gly Ser Pro Cys Pro Glu Leu Glu 305 310 315 320 Glu Glu Ala Glu CysVal Pro Asp Asn Cys Val 325 330 3 19 DNA artificial sequence primer 3cgcgcatagc tccgactac 19 4 15 DNA artificial sequence primer 4 gccgcgtccgcaaag 15 5 30 DNA artificial sequence probe 5 aggaagaacc agtacgtcagtaacgggctg 30 6 36 DNA artificial sequence primer 6 tccctctagagccaccatgg aaaaccccag cccggc 36 7 35 DNA artificial sequence primer2 7aaggcatcac gtgttagacg cagttatcag ggacg 35 8 19 PRT Homo sapiens 8 ProLeu Gly Gly Glu Ser Ile Cys Ser Ala Gly Ala Pro Ala Lys Tyr 1 5 10 15Ser Ile Thr 9 19 PRT Homo sapiens 9 Thr Phe Thr Gly Lys Trp Ser Gln ThrAla Phe Pro Lys Gln Tyr Pro 1 5 10 15 Leu Phe Arg 10 15 PRT Homo sapiens10 His Ser Ser Asp Tyr Ser Met Trp Arg Lys Asn Gln Tyr Val Ser 1 5 10 1511 23 PRT Homo sapiens 11 Asp Ala Gly Thr Asp Ser Gly Phe Thr Phe SerSer Pro His Phe Ala 1 5 10 15 Thr Ile Pro Gln Asp Thr Val 20 12 12 PRTHomo sapiens 12 Asn Glu Ile Val Asp Ser Ala Ser Val Pro Glu Thr 1 5 1013 330 PRT Rattus norvegicus 13 Met Glu Asn Val Ser Phe Ser Leu Asp ArgThr Leu Trp Val Phe Leu 1 5 10 15 Leu Ala Met Leu Gly Ser Thr Ala GlyGln Pro Leu Gly Gly Glu Ser 20 25 30 Val Cys Thr Ala Arg Pro Leu Ala ArgTyr Ser Ile Thr Phe Thr Gly 35 40 45 Lys Trp Ser Gln Thr Ala Phe Pro LysGln Tyr Pro Leu Phe Arg Pro 50 55 60 Pro Ala Gln Trp Ser Ser Leu Leu GlyAla Ala His Ser Ser Asp Tyr 65 70 75 80 Ser Met Trp Arg Lys Asn Glu TyrVal Ser Asn Gly Leu Arg Asp Phe 85 90 95 Ala Glu Arg Gly Glu Ala Trp AlaLeu Met Lys Glu Ile Glu Ala Ala 100 105 110 Gly Glu Lys Leu Gln Ser ValHis Ala Val Phe Ser Ala Pro Ala Val 115 120 125 Pro Ser Gly Thr Gly GlnThr Ser Ala Glu Leu Glu Val His Pro Arg 130 135 140 His Ser Leu Val SerPhe Val Val Arg Ile Val Pro Ser Pro Asp Trp 145 150 155 160 Phe Val GlyIle Asp Ser Leu Asp Leu Cys Glu Gly Gly Arg Trp Lys 165 170 175 Glu GlnVal Val Leu Asp Leu Tyr Pro His Asp Ala Gly Thr Asp Ser 180 185 190 GlyPhe Thr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp Thr 195 200 205Val Thr Glu Ile Thr Ala Ser Ser Pro Ser His Pro Ala Asn Ser Phe 210 215220 Tyr Tyr Pro Arg Leu Lys Ser Leu Pro Pro Ile Ala Lys Val Thr Phe 225230 235 240 Val Arg Leu Arg Gln Ser Pro Arg Ala Phe Ala Pro Pro Ser LeuAsp 245 250 255 Leu Ala Ser Arg Gly Asn Glu Ile Val Asp Ser Leu Ser ValPro Glu 260 265 270 Thr Pro Leu Asp Cys Glu Val Ser Leu Trp Ser Ser TrpGly Leu Cys 275 280 285 Gly Gly Pro Cys Gly Lys Leu Gly Ala Lys Ser ArgThr Arg Tyr Val 290 295 300 Arg Val Gln Pro Ala Asn Asn Gly Thr Pro CysPro Glu Leu Glu Glu 305 310 315 320 Glu Ala Glu Cys Ala Pro Asp Asn CysVal 325 330

What is claimed is:
 1. A method for detecting metastasis of prostatecancer in a subject, wherein the method comprises: (a) obtaining fromthe subject a non-prostate tissue and/or fluid sample; (b) contactingthe sample with an antibody or antibody fragment which specificallybinds to one or more epitopes present in a human RG1 polypeptide havingthe amino acid sequence of SEQ ID NO: 2; (c) detecting the binding ofthe antibody or antibody fragment with the human RG1 polypeptide in thesample; and (d) determining if the level of binding in the subject isincreased over the level detected in normal controls.
 2. The method ofclaim 1, wherein the antibody or antibody fragment is labeled with acompound selected from the group consisting of a radiolabel, achromophore and a fluorescer, so as to directly or indirectly produce adetectable signal.
 3. The method of claim 1, wherein the antibodyfragment is a F(ab′)2 fragment.
 4. The antibody of claim 1, wherein theantibody or antibody fragment specifically binds to the amino acidsequence PLGGESICSAGAPAKYSIT (SEQ ID NO: 8).
 5. The antibody of claim 1,wherein the antibody or antibody fragment specifically binds to theamino acid sequence HSSDYSMWRKNQYVS (SE ID NO: 10).
 6. The antibody ofclaim 1, wherein the antibody or antibody fragment specifically binds tothe amino acid sequence DAGTDSGFTFSSPNFATIPQDTV (SEQ IDNO: 11).
 7. Theantibody of claim 1, wherein the antibody or antibody fragmentspecifically binds to the amino acid sequence NEIVDSASVPET (SEQ IDNO:12).
 8. The antibody of claim 1, wherein the antibody is a polyclonalantibody.
 9. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 10. A method of detecting metastasis of prostatecancer in a subject, wherein the method comprises: (a) injecting thesubject with an appropriate dose of a radiolabeled antibody orantibody-fragment which specifically binds to one or more epitopespresent in a human RG1 polypeptide having the amino acid sequence of SEQID NO: 2; (b) detecting by immunoscintography the binding of theradiolabeled antibody or antibody fragment to one or more epitopespresent in the human RG1 polypeptide within the subject; and (c)determining if the level of binding in thc subject is increased over thelevel detected in normal controls.
 11. The method of claim 10, whereinthe radiolabel is In-111 or tc-99m.